VEGFA-targeting miR-agshRNAs combine efficacy with specificity and safety for retinal gene therapy

Sidsel Alsing, Thomas Koed Doktor, Anne Louise Askou, Emilie Grarup Jensen, Ulvi Ahmadov, Lasse Sommer Kristensen, Brage Storstein Andresen, Lars Aagaard, Thomas J. Corydon*

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Abstract

Retinal gene therapy using RNA interference (RNAi) to silence targeted genes requires both efficacy and safety. Short hairpin RNAs (shRNAs) are useful for RNAi, but high expression levels and activity from the co-delivered passenger strand may cause undesirable cellular responses. Ago2-dependent shRNAs (agshRNAs) produce no passenger strand activity. To enhance efficacy and to investigate improvements in safety, we have generated VEGFA-targeting agshRNAs and microRNA (miRNA)-embedded agshRNAs (miR-agshRNAs) and inserted these RNAi effectors in Pol II/III-driven expression cassettes and lentiviral vectors (LVs). Compared with corresponding shRNAs, agshRNAs and miR-agshRNAs increased specificity and safety, while retaining a high knockdown efficacy and abolishing passenger strand activity. The agshRNAs also caused significantly smaller reductions in cell viability and reduced competition with the processing of endogenous miR21 compared with their shRNA counterparts. RNA sequencing (RNA-seq) analysis of LV-transduced ARPE19 cells revealed that expression of shRNAs in general leads to more changes in gene expression levels compared with their agshRNA counterparts and activation of immune-related pathways. In mice, subretinal delivery of LVs encoding tissue-specific miR-agshRNAs resulted in retinal pigment epithelium (RPE)-restricted expression and significant knockdown of Vegfa in transduced RPE cells. Collectively, our data suggest that agshRNAs and miR-agshRNA possess important advantages over shRNAs, thereby posing a clinically relevant approach with respect to efficacy, specificity, and safety.

OriginalsprogEngelsk
TidsskriftMolecular Therapy - Nucleic Acids
Vol/bind28
Sider (fra-til)58-76
Antal sider19
ISSN2162-2531
DOI
StatusUdgivet - 14. jun. 2022

Bibliografisk note

Funding Information:
The authors would like to thank Tina Hindkjaer and Kamilla Zahll Hornbek for their excellent technical support. Flow cytometry analysis and FACS were performed at the FACS Core Facility, Aarhus University, Denmark, and the authors would like to thank staff at the core facility for their support. Paraffin embedding and sectioning were performed at the Department of Pathology, Aarhus University Hospital. This work was supported by the Faculty of Health Sciences, Aarhus University (PhD scholarship to S.A.), the Danish Eye Research Foundation (T.J.C.), Aase and Ejnar Danielsen's Foundation (T.J.C.), Bagenkop-Nielsens Myopifond, Svend Helge Schr?der og hustru Ketty Lydia Larsen Schr?ders fond (T.J.C.), Maskinfabrikant Jochum Jensen og hustru Mette Marie Jensen F. Poulsens Mindelegat (S.A.), and The Velux Foundation (T.J.C.). Writing ? original draft, S.A. A.L.A. L.A. and T.J.C.; writing ? reviewing & editing, S.A. A.L.A. E.G.J. T.K.D. U.A. L.S. B.S.A, L.A. and T.J.C.; supervision, T.J.C. L.A. L.S. B.S.A. and A.L.A.; investigation, S.A. T.K.D. E.G.J. U.A. and A.L.A.; methodology, S.A. T.K.D. L.A. T.J.C. A.L.A. L.S. and B.S.A. The authors declare no competing interests.

Funding Information:
The authors would like to thank Tina Hindkjaer and Kamilla Zahll Hornbek for their excellent technical support. Flow cytometry analysis and FACS were performed at the FACS Core Facility, Aarhus University, Denmark, and the authors would like to thank staff at the core facility for their support. Paraffin embedding and sectioning were performed at the Department of Pathology, Aarhus University Hospital. This work was supported by the Faculty of Health Sciences, Aarhus University (PhD scholarship to S.A.), the Danish Eye Research Foundation (T.J.C.), Aase and Ejnar Danielsen’s Foundation (T.J.C.), Bagenkop-Nielsens Myopifond, Svend Helge Schrøder og hustru Ketty Lydia Larsen Schrøders fond (T.J.C.), Maskinfabrikant Jochum Jensen og hustru Mette Marie Jensen F. Poulsens Mindelegat (S.A.), and The Velux Foundation (T.J.C.).

Publisher Copyright:
© 2022 The Authors

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